Low numerical aperture lens based oblique plane illumination imagining

ABSTRACT

An imaging system includes a first finite conjugate objective at a frontal end of the system and a second finite conjugate objective at a distal end of the system. The system also includes a beam splitting or merging element positioned between the first finite conjugate objective and the second finite conjugate objective. The system also includes an excitation unit configured to direct an excitation beam into a sample positioned in front of the first finite conjugate objective. The excitation beam is in the form of an excitation plane. The system also includes an image sensor positioned facing the second finite conjugate objective. The image sensor lies in a conjugate plane of an excitation beam illumination plane at the frontal end of the system.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the priority benefit of U.S. ProvisionalPatent App. No. 62/797,997 filed on Jan. 29, 2019, the entire disclosureof which is incorporated by reference herein.

BACKGROUND

Single front-facing microscope objective based oblique planeillumination imaging and microscopy is a powerful imaging technique thatallows for steric access to the sample being imaged. Traditionalobjective based oblique plane illumination image systems typicallyinclude three high numerical aperture (NA) objectives placedsequentially along the imaging path, where last two objectives areplaced at an angle to each other to enable imaging of the obliqueintermediate image.

SUMMARY

An illustrative imaging system includes a first finite conjugateobjective at a frontal end of the system and a second finite conjugateobjective at a distal end of the system. The system also includes a beamsplitting or merging element positioned between the first finiteconjugate objective and the second finite conjugate objective. Thesystem also includes an excitation unit configured to direct anexcitation beam into a sample positioned in front of the first finiteconjugate objective. The excitation beam is in the form of an excitationplane. The system also includes an image sensor positioned facing thesecond finite conjugate objective. The image sensor lies in a conjugateplane of an excitation beam illumination plane at the frontal end of thesystem.

In an illustrative embodiment, the first finite conjugate objective andthe second finite conjugate objective have a low numerical aperture.Also, the beam splitting or merging element can be a cube beam splitteror a plate beam splitter with excitation and emission filters. The beamsplitting or merging element can also be a dichroic beam splitter whichallows only a fluorescence emission beam to pass through to the distalend of the system. The excitation unit can include any combination of alaser or laser diode, a beam expander, a slit aperture, and acylindrical lens to shape the excitation beam into a planar excitationbeam. Alternatively, the excitation unit can include any combination ofa laser, a galvanometer mounted planar-mirror based scanner, and aconverging lens to form a planar excitation beam. The excitation unitemits the excitation beam at an inclined angle such that resultantillumination is an oblique plane, tilted with respect to a principalaxis of the first finite conjugate objective. A tilt angle of theexcitation beam and the image sensor can be matched such that the imagesensor is able to image an entire illuminated plane at the same time.

The image sensor can be a two-dimensional (2D) light detector arraywhich acquires 2D images through a global shutter mechanism or through arolling shutter mechanism while the image sensor is staticallypositioned. The system can also include a translation stage to hold thesample, where the translation stage moves the sample in a directionperpendicular to the principal axis of the first finite conjugateobjective such that the sample can be moved to form an image fromneighboring optical slices which can be stitched together to form athree-dimensional image of the sample. The system can also include acomputing system configured to apply an Affine transformation on a stackof acquired images, where application of the Affine transformation isbased on a tilt angle of the excitation plane.

An imaging system in accordance with a second embodiment includes amagnifying unit positioned at a frontal end of the system, where themagnifying unit includes a first infinity corrected objective and afirst tube lens. The system also includes a de-magnifying unitpositioned at a distal end of the system, where the de-magnifying unitincludes a second infinity corrected objective and a second tube lens.The system also includes a beam splitting or merging element positionedin between the first infinity corrected objective and the secondinfinity corrected objective. The system also includes an excitationunit configured to direct an excitation beam into a sample positioned infront of the first infinity corrected objective, where the excitationbeam is in the form of an excitation plane. The system also includes animage sensor positioned directly in front of the second infinitycorrected objective such that the image sensor lies in a conjugate planeof the excitation plane.

In an illustrative embodiment, the first infinity corrected objectiveand the second infinity corrected objective have a low numericalaperture. Also, the beam splitting or merging element can be a cube beamsplitter or a plate beam splitter with excitation and emission filters.Alternatively, the beam splitting or merging element can be a dichroicbeam splitter which allows only a fluorescence emission beam to passthrough to the distal end of the system. The beam splitting or mergingelement is positioned between the first infinity corrected objective andthe first tube lens. Alternatively, the beam splitting or mergingelement is positioned between the first tube lens and the second tubelens. In another alternative embodiment, the beam splitting or mergingelement is positioned between the second tube lens and the secondinfinity corrected objective.

The excitation unit of the system can include any combination of a laseror laser diode, a beam expander, a slit aperture and a cylindrical lensto shape the excitation beam into a planar excitation beam.Alternatively, the excitation unit can include any combination of alaser, a galvanometer mounted planar-mirror based scanner, and aconverging lens to form a planar excitation beam. The excitation unitemits the excitation beam either from an off-axis position or from aninclined position such that resultant illumination is an oblique planethat is tilted with respect to a principal axis of the first infinitycorrected objective. Tilt angles of the excitation plane and the imagesensor are matched such that the image sensor can image an entireportion of the excitation plane at the same time. The image sensor ofthe system can be a two-dimensional (2D) light detector array whichacquires 2D images either through a global shutter mechanism or througha rolling shutter mechanism while the image sensor is staticallypositioned. The system can also include a translation stage that movesthe sample in a direction perpendicular to an axis of the first infinitycorrected objective such that the sample can be moved to form an imagefrom neighboring optical slices which can be stitched together to form athree-dimensional image of the sample. The system can also include acomputing system configured to apply an Affine transformation on a stackof acquired images to obtain an undistorted three-dimensionalreconstruction of the sample, where application of the Affinetransformation is based on a tilt angle of the excitation plane.

An imaging system in accordance with a third illustrative embodimentincludes a magnifying unit at a frontal end of the system, where themagnifying unit includes a first infinity corrected objective and afirst tube lens. The system also includes a de-magnifying unit at adistal end of the system, where the de-magnifying unit includes a secondinfinity corrected objective and a second tube lens. The system includesa scanning unit positioned between the magnifying unit and thede-magnifying unit, and a beam splitting or merging element positionedbetween the second tube lens and the second infinity correctedobjective. The system also includes an excitation unit configured toemit an excitation beam into a sample positioned in front of the firstinfinity corrected objective, where the excitation beam is in the formof an excitation plane. The system further includes an image sensorpositioned directly in front of the second infinity corrected objectivesuch that the image sensor lies in a conjugate plane of the excitationplane.

In an illustrative embodiment, the first infinity corrected objectiveand the second infinity corrected objective have a low numericalaperture. The scanning unit can include a galvanometer mounted planarmirror positioned between two converging lenses such that an axis ofrotation of the galvanometer mounted planar mirror matches focal planesof the two converging lenses. The magnifying unit at the frontal end,the scanning unit, and the de-magnifying unit at the distal end arestacked together sequentially to provide a net magnification which isthe same along a lateral direction and an axial direction, and where thenet magnification has a numerical value between 1 and 2. In oneembodiment, the beam splitting or merging element includes a cube beamsplitter or a plate beam splitter with excitation and emission filters.Alternatively, the beam splitting or merging element includes a dichroicbeam splitter which allows only a fluorescence emission beam to passthrough to the distal end of the system, and the beam splitting ormerging element can be positioned between the tube lens and the secondinfinity corrected objective at the distal end of the system.

The excitation unit includes any combination of a laser or laser diode,a beam expander, a slit aperture, and a cylindrical lens to shape theexcitation beam into a planar excitation beam. Alternatively, theexcitation unit includes any combination of a laser, a galvanometermounted planar-mirror based scanner, and a converging lens to form aplanar excitation beam. The excitation unit emits the excitation beamfrom an off-axis position such that resultant illumination is an obliqueplane tilted with respect to a principal axis of the first infinitycorrected objective. Tilt angles of the excitation plane and the imagesensor match such that the image sensor can image the entire excitationplane at the same time. The image sensor can be a two-dimensional (2D)light detector array that acquires 2D images either through a globalshutter mechanism or through a rolling shutter mechanism withoutmovement of the image sensor. Also, rotation of a planar mirror of thescanning unit gives rise to a constant tilt lateral shift in theexcitation plane, and a de-scan of a received signal beam results in astatic imaging plane at the distal end of the system such that thesample can be imaged while stationary. The system can also include acomputing system configured to apply an Affine transformation on a stackof acquired images to obtain an undistorted three-dimensionalreconstruction of the sample, where application of the Affinetransformation is based on the tilt angle of the excitation plane.

An imaging system in accordance with a fourth illustrative embodimentincludes a first set of optical elements including a first infinitycorrected objective positioned at a frontal end of the system, a beamsplitting or merging unit, an image relay and scan unit, and a secondinfinity corrected objective positioned at a distal end of the system.The system also includes a second set of optical elements including athird infinity corrected objective, a tube lens, and an image sensor.The system also includes a diffusive screen positioned between the firstset of optical elements and the second set of optical elements. Thesystem further includes an excitation unit configured to emit anexcitation beam into a sample positioned along an axis of the beamsplitting or merging unit, where the excitation beam is in the form of aplanar excitation beam.

In an illustrative embodiment, a combination the first infinitycorrected objective, the second infinity corrected objective, and thethird infinity corrected objective has a low numerical aperture. Also,the image relay and scan unit can include a galvanometer mounted planarmirror positioned between two converging lenses such that an axis ofrotation of the galvanometer mounted planar mirror exactly matches withfocal planes of the two converging lenses. The first set of opticalelements can be positioned between the sample and the diffusive screento provide a net magnification which is the same along a lateraldirection and an axial direction, and the net magnification can have anumerical value between 1 and 2. The diffusive screen acts as aprojection screen and is made of scattering particles, where a size ofthe scattering particles either smaller than a resolving power of thesystem or is comparable to the resolving power of the system. In someembodiments, the diffusive screen is mounted on a moving rotor orvibration motor which allows for in-plane motion of the scatteringparticles to average out random particle images that overlay images ofthe sample.

The second set of optical elements form on the image sensor a magnifiedview of an intermediate image at the diffusive screen to allow for useof a large format image sensor. The beam splitting or merging unit caninclude a cube beam splitter or a plate beam splitter with excitationand emission filters in one embodiment. Alternatively, the beamsplitting or merging unit includes a dichroic beam splitter that allowsonly a fluorescence emission beam to pass through to the distal end ofthe system, and the beam splitting or merging unit can be positionedbetween the tube lens and the third infinity corrected objective. Inanother embodiment, the beam splitting or merging unit includes a cubebeam splitter, a plate beam splitter, or a dichroic mirror, and afluorescence filter positioned between the diffusive screen and theimage sensor to enable exact selection of a fluorescence emission.

In one embodiment, the excitation unit includes any combination of alaser or laser diode, a beam expander, a slit aperture, and acylindrical lens to shape the excitation beam into the planar excitationbeam. Alternatively, the excitation unit includes any combination of alaser, a galvanometer mounted planar-mirror based scanner, and aconverging lens to form the planar excitation beam. The excitation unitemits the excitation beam from an off-axis position such that resultantillumination is an oblique plane tilted with respect to a principal axisof the first infinity corrected objective. A tilt angle of planarexcitation beam, diffusive screen, and image sensor are exactly matchedsuch that the image sensor is able to image an entire illuminated planeat the same time. The image sensor can be a two-dimensional (2D) lightdetector array which acquires 2D images either through a global shuttermechanism or through a rolling shutter mechanism without movement of theimage sensor. Also, rotation of a planar mirror of the image relay andscan unit gives rise to a constant tilt lateral shift in an illuminatingoblique plane, and a de-scan of a received signal beam results in astatic imaging plane at the diffusive screen and on the image sensorwithout translation of the sample. The system can also include acomputing system configured to apply an Affine transformation on a stackof acquired images to obtain an undistorted three-dimensionalreconstruction of the sample, where application of the affinetransformation is based on a tilt angle of the excitation plane.

Other principal features and advantages of the invention will becomeapparent to those skilled in the art upon review of the followingdrawings, the detailed description, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments of the invention will hereafter be describedwith reference to the accompanying drawings, wherein like numeralsdenote like elements.

FIG. 1A depicts a conventional oblique plane imaging setup.

FIG. 1B is a partial view of a conventional oblique plane imaging setupthat shows an acceptance cone between a second microscope objective anda third microscope objective of the system.

FIG. 2 is a table that provides an estimate to net system numericalaperture (NA) and corresponding theoretical resolution for variouschoices of objectives in an oblique plane illumination imaging system.

FIG. 3 depicts a low NA objective based oblique plane illuminationimaging system in accordance with an illustrative embodiment.

FIG. 4 depicts the effective NA of the system of FIG. 3 in accordancewith an illustrative embodiment.

FIG. 5A depicts a top view of an illumination sub-system in accordancewith an illustrative embodiment.

FIG. 5B depicts a side view of the illumination sub-system of FIG. 5A inaccordance with an illustrative embodiment.

FIG. 6A depicts a top view of an illumination sub-system in accordancewith an illustrative embodiment.

FIG. 6B depicts a side view of the illumination sub-system of FIG. 6A inaccordance with an illustrative embodiment.

FIG. 7 depicts a side view of an illumination sub-system in accordancewith an illustrative embodiment.

FIG. 8 depicts a low NA lens based oblique plane illumination imagingsystem in accordance with a first illustrative embodiment.

FIG. 9 depicts a low NA lens based oblique plane illumination imagingsystem in accordance with a second illustrative embodiment.

FIG. 10 depicts a low NA lens based oblique plane illumination imagingsystem in accordance with a third illustrative embodiment.

FIG. 11 depicts a low NA lens based oblique plane illumination imagingsystem in accordance with a fourth illustrative embodiment.

FIG. 12 is a block diagram of a computing device in communication with anetwork in accordance with an illustrative embodiment.

FIG. 13 depicts a United States Air Force (USAF) resolution test targetimaged in brightfield illumination using the oblique plane illuminationimaging system of FIG. 8 in accordance with an illustrative embodiment.

FIG. 14 depicts a USAF resolution test target imaged in brightfieldillumination using the oblique plane illumination imaging system of FIG.11 in accordance with an illustrative embodiment.

DETAILED DESCRIPTION

Existing oblique plane imaging approaches use sequential arrangements ofthree high numerical aperture (NA) microscope objectives, leading todramatic loss in NA from the oblique arrangement of last two objectives(i.e., a distal end of the microscope away from the sample beingimaged). This leads to two major limitations: (1) low effective NA ofthe entire imaging system, and (2) restricting this class of light-sheetimaging techniques to high NA objectives alone (to partly compensate forthe loss of NA). Described herein are methods and systems that overcomethese limitations and that are able to perform oblique plane imagingwith a selection of low NA objectives.

FIG. 1A depicts a conventional oblique plane imaging setup. The obliqueplane imaging setup includes a first microscope objective 105, an imagerelay and scan unit 110, a second microscope objective 115, a thirdmicroscope objective 120, and a tube lens and camera 125. As shown, thefirst microscope objective 105 is proximate to a point object 100. FIG.1B is a partial view of a conventional oblique plane imaging setup thatshows an acceptance cone between the second microscope objective 115 andthe third microscope objective 120. As shown in FIG. 1B, a netacceptance cone 130 of the system is smaller than the overall cone 135,which results in a loss in net NA for the system. This loss arises dueto the oblique placement of the second microscope objective 115 and thethird microscope objective 120 in the conventional system.

Solutions to the aforementioned technical problems with conventionaloblique plane imaging systems can be found by approaches describedherein, including: (1) removing the third objective from the obliqueplane imaging systems and placing the image sensor directly at theintermediate image plane in front of second objective and/or (2) using adiffusive screen at the intermediate image plane after the secondobjective, and then using a third objective based magnifying system tore-image the illuminated sample plane on an image sensor. Both of thesesolutions are no longer restrictive in terms of objective NA and workwith low NA objectives.

In one of the embodiments described herein, the third objective isremoved from the oblique plane imaging system. The omission of oneobjective and corresponding supporting optics makes it possible toreduce the system size, and thus miniaturization of the whole system isachievable. In all of the embodiments described herein, the choice oflow NA objectives reduces the overall system cost while still retainingthe advantageous effects of oblique plane imaging which include stericaccess to the sample being imaged, a single front facing objective basedlight-sheet architecture, constant tilt scanning of an illuminatingplanar excitation beam, leading to true perspective three dimensional(3D) reconstruction of the scanned sample, etc.

Oblique plane imaging and microscopy is an approach for making use of asingle front facing objective based setup to perform light-sheetmicroscopy. In traditional systems (e.g., FIG. 1A), an off-axis beamincident on the main objective provides an oblique illumination plane inthe sample, the second objective forms an intermediate image of theoblique illumination plane, and the inclined third objective correctsfor the tilt and forms a magnified final image on the image sensor.Variants of this oblique plane imaging setup have been suggested whicheither make use of an alternative illumination or scanning architectureto perform rapid 3D imaging of a sample. All of the existing variants ofoblique plane imaging setups include the components as described in FIG.1A.

As seen from FIGS. 1A and 1B, there is a dramatic loss in the netacceptance angle of the system at the interface between second and thirdobjectives of the system. This inclined placement of third objective hastraditionally been used because any attempt on increasing the system NAby co-aligning the third objective with the second objective would throwmost of the image plane out of focus, with only the center line of theimage remaining in focus on the image sensor. Due to this loss in systemNA, it becomes mandatory in traditional systems to use high NAobjectives in the oblique plane imaging setup. FIG. 2 is a table thatprovides an estimate to net system NA and corresponding theoreticalresolution for various choices of objectives. For simplicity, all threeobjectives of the oblique plane imaging system are assumed to be of thesame NA, and the wavelength of light is assumed to be 520 nanometers(nm), which is the central wavelength for most widely used fluorescentmarkers in biology (i.e. green fluorescent protein (GFP) and itsderivatives). For system NA=0, the oblique plane imaging setup fails,and hence system resolution numbers are not included in the table.

Having the knowledge of numerical aperture (first column of the table inFIG. 2) of an objective (or imaging system), Rayleigh criterion helpsdetermine its resolution (second column of the table) by the followingrelationship:

$\begin{matrix}{{Resolution} = {0.61 \times \frac{\lambda}{NA}}} & {{Eq}.\mspace{14mu} 1}\end{matrix}$

In Equation 1, ‘λ’ refers to the wavelength of light used. Thedefinition of numerical aperture (NA) helps in determining the maximumacceptance angle and hence the maximum tilt angle of the oblique planeillumination (third column of the table) by the following relation:

$\begin{matrix}{{\varphi = {\sin^{- 1}\left( \frac{NA}{n} \right)}},} & {{Eq}.\mspace{14mu} 2}\end{matrix}$

In Equation 2, ‘φ’ is the maximum tilt angle and ‘n’ is the refractiveindex of immersion media of the given objective (n=1.33 for thegeneralized case depicted in the table of FIG. 2). In alternativeembodiments, a different refractive index of immersion media may beused. The effective NA of the oblique plane imaging system (fourthcolumn of the table in FIG. 2) is determined by calculating theeffective angle of overlap between acceptance angles of the second andthird objectives, which is (3φ−90°). A negative value of this angleimplies that there is no overlap and hence system NA is zero. Forpositive values, the system NA is given by sin(1.5φ−45°). Finally, thesystem resolution (fifth column of the table) is obtained by using theRayleigh resolution relationship of Equation 1 with the determinedsystem NA value.

It is evident from the table of FIG. 2 that conventional oblique planeimaging techniques work only for high NA objective choices. Even with amoderately high NA of 0.5, the system fails to image. The aboveconsiderations are general, and in practice it is possible to mix thechoice of objectives such that the amount of overlap between the secondand third objectives is optimized, even with one of them being a high NAobjective. Still, the table illustrates the strong dependence of thisclass of imaging techniques on NA, and it is impossible to image withe.g., 0.3 NA or smaller objectives.

From the table of FIG. 2, it can be seen that NA=0.3 objectives areindividually capable of ˜1 micrometer (μm) resolution, but fail toresolve anything when used in a conventional oblique plane imagingsetup. At the same time, it is noted that the loss in effective NA of anoblique plane imaging setup comes at the interface of the second andthird objective. The novel solutions described herein use a low NAobjective in oblique plane imaging to get rid of the interface where NAloss occurs. Specifically, in some embodiments, the third objective andrelated optics are removed, and are replaced by a small pixel imagesensor positioned directly at the intermediate plane ahead of the secondobjective.

FIG. 3 depicts a low NA objective based oblique plane illuminationimaging system in accordance with an illustrative embodiment. As shown,the oblique plane imaging system includes a first microscope objective300, an image relay and scan unit 305, an excitation unit 310, and asecond microscope objective 315. Also depicted in FIG. 3 are anillumination plane 320 and an image plane 325. In the embodiment of FIG.3, a small pixel image sensor is placed directly at the intermediateimage plane 325 ahead of the second microscope objective 315. The imagesensor can be a camera or other image capturing device. The embodimentof FIG. 3 allows most of the NA of the system to be retained.Theoretically, the system NA could be the same as that of individualobjectives. However, in practice there is a small loss in NA due to theimage sensor placement being at a smaller angle (with respect to theaxis of the objective) than that dictated by the objective NA. This isdone to avoid clipping of the finite aperture beam at the edge of theobjective, and it leads to a slight reduction of acceptance angle asshown in FIG. 4.

FIG. 4 depicts the effective NA of the system of FIG. 3 in accordancewith an illustrative embodiment. The effective NA is due to directplacement of an image sensor on the intermediate image plane ahead ofthe second microscope objective 315. FIG. 4 shows an acceptance cone 400of the second microscope objective 315, a net acceptance cone 405, andan image sensor plane 410. Also shown is the effective acceptance angle415 (i.e., the darker shaded region).

In the embodiment depicted in FIGS. 3 and 4, the system NA (˜0.3)leading to ˜1 μm attainable resolution is no longer the limiting factorwhen imaging most biological samples at the cellular level. However, thesystem resolution is now limited by the image sensor pixel size. Forthis reason, small pixel image sensors (e.g., sensors finding wideapplications in modern mobile phones) are both a low-cost and efficientchoice for the proposed system. As an example, the Sony IMX219PQ sensorwith 1.12 μm pixel size and easy availability is a good fit for theproposed system. In alternative embodiments, a different type and/orsize image sensor may be used.

The effective pixel size in the sample plane depends on themagnification factor between the sample plane and the image sensorplane. For the case where both objectives are dry, this magnificationfactor is unity. For another case where the first objective is a waterimmersion type, followed by a dry second objective, the magnificationfactor is 1.33, thus further improving the attainable resolution due tothe smaller effective pixel size (1.12/1.33=0.92 μm) in the sampleplane. The magnification factor of 1.33 in the case of a water-dryobjective combination arises due to the desire to maintain equal lateraland axial magnification of the system between sample and image planes,so that the images captured by sensors are undistorted, whichstreamlines the three-dimensional (3D) reconstruction of samples.

Described in detail below are four embodiments of oblique planeillumination imaging systems that utilize low NA objectives. Additionalembodiments are also possible using the techniques and systems describedherein. In a first embodiment, the system uses two finite conjugateobjectives to form a static oblique illumination plane. In a secondembodiment, the system includes two infinity corrected objectives, alongwith a matching tube-lens, for forming a static oblique illuminationplane. In the first and second embodiments, a 3D image can be capturedby mounting the sample on a translation stage that is able to repositionthe sample during the imaging process. In a third embodiment, twoinfinity corrected objectives are used along with a matching tube-lensand a scan unit, which helps form a constant tilt scanned oblique planeillumination plane. As such, in the third embodiment, a 3D image can becaptured without translating the sample. In a fourth embodiment, thesystem avoids use of a small pixel image sensor by incorporating adiffusive screen at the intermediate image plane. In this embodiment,the system re-images the screen projected image with a third objectiveand a tube lens assisted magnification unit, which forms the final imageon a regular scientific image sensor.

The embodiments described herein include an imaging sub-system, anillumination sub-system, a scanning sub-system, and a 3D reconstructionsub-system/method. Among the sub-systems, the illumination sub-system isresponsible for the creation of a planar illumination beam. Theillumination sub-system can be implemented in a number of differentways, several of which are described below.

FIG. 5A depicts a top view of an illumination sub-system in accordancewith an illustrative embodiment. FIG. 5B depicts a side view of theillumination sub-system of FIG. 5A in accordance with an illustrativeembodiment. The embodiment of FIGS. 5A and 5B includes a laser diode500, a collimating lens 505, s slit aperture 510, and a cylindrical lens515. In alternative embodiments, the sub-system may include fewer,additional, and/or different components. In the embodiment of FIGS. 5Aand 5B, a diverging beam 520 from the laser diode 500 is collimatedusing the collimating lens 505 and then passed through the slit aperture510 and the cylindrical lens 515 to form a planar illumination beam 525.

FIG. 6A depicts a top view of an alternative illumination sub-system inaccordance with an illustrative embodiment. FIG. 6B depicts a side viewof the illumination sub-system of FIG. 6A in accordance with anillustrative embodiment. The embodiment of FIGS. 6A and 6B includes alaser 600, a beam expander 605, a slit aperture 610, and a cylindricallens 615. In alternative embodiments, the sub-system may include fewer,additional, and/or different components. In the embodiment of FIGS. 6Aand 6B, a collimated laser beam 620 from the laser 600 is expanded bythe beam expander 605, and passed through the slit aperture 610 and thecylindrical lens 615 to form a planar illumination beam 625.

FIG. 7 depicts a side view of an illumination sub-system in accordancewith another illustrative embodiment. The embodiment of FIG. 7 includesa laser 700, a galvo scanner (or galvanometer) 705, and a scan lens 710.In alternative embodiments, the sub-system of FIG. 7 may include fewer,additional, and/or different components. In the embodiment of FIG. 7, alaser beam 715 is scanned through a mounted planar mirror 720 of thegalvanometer 705, and then focused through the scan lens 710 to form aplanar illumination beam 725.

In an illustrative embodiment, the choice of source wavelength in theillumination sub-system (i.e., any of the sub-systems depicted in FIGS.5-7) is based on the fluorescent protein or dye of interest. Also, insome embodiments, multiple illumination sources may be used to allow formulti-color imaging. In such an embodiment, different types ofillumination sources may be used.

In another illustrative embodiment, the 3D reconstructionsub-system/method described herein can be common among all of thedescribed embodiments. The 3D dataset is acquired by a tilted planarillumination, and therefore deviates from a conventional datasetacquired in Cartesian coordinates. As a result, the 3D dataset isgeometrically skewed and a 3D Affine transformation is utilized tocorrect the orientation. The Affine transformation can be a combinationof two geometrical transforms: scale and shear, and is given by thefollowing matrix:

$\begin{matrix}{{{{M_{sh} \times M_{sc}} = {{\begin{bmatrix}1 & 0 & 0 & 0 \\0 & 1 & 0 & 0 \\0 & {- {tan\theta}} & 1 & 0 \\0 & 0 & 0 & 1\end{bmatrix} \times \begin{bmatrix}1 & 0 & 0 & 0 \\0 & {cos\theta} & 0 & 0 \\0 & 0 & 1 & 0 \\0 & 0 & 0 & 1\end{bmatrix}} =}}\quad}{\quad\begin{bmatrix}1 & 0 & 0 & 0 \\0 & {cos\theta} & 0 & 0 \\0 & {- {sin\theta}} & 1 & 0 \\0 & 0 & 0 & 1\end{bmatrix}}} & {{Eq}.\mspace{14mu} 3}\end{matrix}$

In the matrix of Eq. 3, ‘0’ is the tilt angle the planar illuminationbeam makes with the principal axis of the first objective. The matrixcan be solved by a computing sub-system that is incorporated into theimaging system or remote from the imaging system, depending on theimplementation. A computing sub-system is described in more detail belowwith reference to FIG. 12.

The description now turns to a detailed discussion of the variousdifferent embodiments referenced above. FIG. 8 depicts a low NA lensbased oblique plane illumination imaging system in accordance with thefirst illustrative embodiment. The first embodiment can be implementedas a compact setup which provides a static oblique plane illuminationinside a given sample. The system of FIG. 8 includes an imagingsub-system that includes a first low NA finite conjugate objective 800,a second low NA finite conjugate objective 805, and a small pixel camerasensor 810. As used herein, low NA can refer to an NA that is smallerthan 0.5. In alternative implementations, low NA can refer to anothervalue, such as smaller than 0.3, smaller than 0.4, smaller than 0.6,etc. As used herein, ‘small pixel’ can refer to a pixel value of smallerthan 5 μm. In alternative implementations, small pixel can refer toanother value, such as smaller than 3 μm, smaller than 4 μm, smallerthan 6 μm, etc.

The system of FIG. 8 also includes an illumination sub-system thatincludes an excitation unit 815 and a beam splitter 820. The excitationunit 815 can include a fluorescence excitation source in the form of alaser or laser diode. The excitation unit 815 can also include a beamexpander, slit aperture, and cylindrical lens, such as that depicted inFIGS. 6A and 6B. Alternatively, instead of the beam expander, slitaperture, and cylindrical lens, the excitation unit 815 may include agalvo scanner and converging lens such as the embodiment depicted inFIG. 7. The beam splitter 820 is used to combine or direct theexcitation beam into the first low NA finite conjugate objective 800 toenable the oblique plane imaging.

The system of FIG. 8 also includes a 3D scanning sub-system 825 that caninclude a sample holder and a translation stage. The translation stageis used to move along the X-Y plane to sweep across a large sample. Asdepicted, an illumination plane 830 is directed onto the 3D scanningsub-system 825. In the embodiment of FIG. 8, 3D reconstruction can beperformed by a stitching algorithm which is used to stitch large 3D scantiles, in conjunction with an Affine transformation which is used toobtain the correct geometrical orientation of the sample. The 3Dreconstruction can be performed by a local or remote computingsub-system that is in direct or indirect communication with the system.In alternative embodiments, the system of FIG. 8 may include fewer,additional, and/or different components.

In the embodiment of FIG. 8, the imaging sub-system is responsible forforming the image of the oblique illumination plane on the small pixelcamera sensor 810. As discussed above, the imaging sub-system includesthe first and second low NA finite conjugate objectives (800, 805) andthe small pixel camera sensor 810. The first and second low NA finiteconjugate objectives (800, 805) are arranged facing away from each otherin such a way that they have a common conjugate image plane. The smallpixel image sensor 810 is kept in proximity to the low NA finiteconjugate objective 805 at the distal end in an oblique orientationwhich precisely matches the tilt of the oblique illumination plane. Thetilts are matched because the net magnification of this imaging system,which is equal to unity, is the same along the lateral direction and theaxial direction.

The oblique plane illumination sub-system 815 has been described abovein detail. The generated planar illumination beam is positioned at theconjugate image plane of the (front facing) low NA finite conjugateobjective 800 with an off-axis tilt. The beam splitter 820 helps directthe light-sheet at an off-axis angle into the objective 800 to form anoblique illumination plane in a sample volume. This illuminated plane isimaged by the symmetrical unity magnification optical system (i.e.,imaging sub-system) at the distal end of the system. The small pixel(and form factor) image sensor 810 can be placed matching the imageplane at the distal end of the system to image the illuminated plane asshown in the FIG. 8.

In the embodiment of FIG. 8, if a fluorescently tagged biological sampleis to be imaged, then the choice of excitation source wavelength anddichroic beam splitter allows for imaging of fluorescent protein, dye,or other sample. This arrangement provides a static planar illuminationand hence images a 2D section inside a given sample. An image sensor canrely on either a global shutter mechanism or a rolling shutter mechanismwithout a need for the sensor to be physically shifted. For 3D imaging,the sample can be mounted on an automated translation stage andsynchronized imaging which registers the sample/stage position can beperformed. Such position-tagged images can then be stitched together toform big volume datasets. Because of the oblique nature of theillumination, the reconstructed volume is skewed. However, a trueorientation reconstruction can be obtained by a simple 3D Affinetransformation operation as described above.

FIG. 9 depicts a low NA lens based oblique plane illumination imagingsystem in accordance with the second illustrative embodiment. The systemof FIG. 9 has an imaging sub-system that includes a first low NAinfinite conjugate (or infinity corrected) objective 900, a second lowNA infinite conjugate (or infinity corrected) objective 905, a firsttube lens 910, a second tube lens 915, and a small pixel camera sensor920. The system also includes an illumination sub-system that includesan excitation unit 925 and a beam splitter 930. The excitation unit 925includes a fluorescence excitation source, which can be in the form of alaser or laser diode. The excitation unit 925 also includes a beamexpander, slit aperture, and cylindrical lens (e.g., as shown in FIGS.6A and 6B), or alternatively a galvo scanner and converging lens (e.g.,as shown in FIG. 7). The beam splitter 930 is used to combine or directthe excitation beam from the excitation unit 925 onto the first low NAinfinite conjugate objective 900 to facilitate the oblique planeimaging.

The system of FIG. 9 also includes a 3D scanning sub-system 935 that caninclude a sample holder and a translation stage. The translation stageis used to move along the X-Y plane to sweep across a large sample. Asdepicted, an illumination plane 940 is directed onto the 3D scanningsub-system 935. In alternative embodiments, the system of FIG. 9 mayinclude fewer, additional, and/or different components.

In the embodiment of FIG. 9, the imaging sub-system is responsible forforming the image of the oblique illumination plane onto the small pixelcamera sensor 920. As discussed, the imaging sub-system includes thefirst and second low NA infinity conjugate objectives (900, 905), thefirst and second tube lenses (910, 915), and the small pixel camerasensor 920. The objective-tube lens pairs are arranged facing away fromeach other in such a way that they have a common conjugate image plane.The small pixel image sensor 920 is kept in proximity to the second lowNA infinity conjugate objective at the distal end of the system in anoblique orientation which gets precisely matched with the tilt of theoblique illumination plane. The tilts are matched because the netmagnification of this imaging system is the same along the lateral andaxial direction (equal to unity) when both objectives are dry.

For some samples, at least one of the objectives in FIG. 9 can be awater immersion objective. In such a case, the magnification ismaintained to be a ratio of indices of water and air, (i.e., 1.33). Thegenerated planar illumination beam is positioned either at the conjugateimage plane of the front facing first low NA infinity correctedobjective 900 with an off-axis tilt (i.e., similar to the firstembodiment) or parallel to the principal axis with an offset at backfocal plane of the objective 900 (i.e., as shown in FIG. 9). The beamsplitter 930 helps direct the beam to form an oblique illumination planein the sample volume. This illuminated plane gets imaged at the distalend of the system. The small pixel (and form factor) image sensor 920 isplaced matching the image plane at the distal end of the system to imagethe illuminated plane, as shown in the FIG. 9. This arrangement providesa static planar illumination and hence images a 2D section inside agiven sample. The effective pixel size in the system is given by camerapixel size/magnification=camera pixel size/1.33 (or camera pixel sizefor all dry objectives). For 3D imaging, the sample can be mounted on anautomated translation stage and a synchronized imaging followed bystitching and 3D Affine transformation can be used to obtain a trueorientation reconstruction as described herein.

FIG. 10 depicts a low NA lens based oblique plane illumination imagingsystem in accordance with the third illustrative embodiment. Asdiscussed in more detail below, the system of FIG. 10 differs from theembodiments of FIGS. 8 and 9 in part because it includes a scan unit for3D microscopy that enables remote, tilt-invariant scanning of theoblique plane illumination. The system of FIG. 10 also includes animaging sub-system that includes a first low NA infinite conjugateobjective 1000, a second low NA infinite conjugate objective 1005, afirst tube lens 1010, a second tube lens 1015, and a small pixel camerasensor 1020.

The illumination sub-system of the embodiment of FIG. 10 includes anexcitation unit 1025 and a beam splitter 1030. The excitation unit 1025includes a fluorescence excitation source (e.g., laser or laser diode).The excitation unit 1025 also includes a beam expander, slit aperture,and cylindrical lens (e.g., as shown in FIGS. 6A and 6B), oralternatively a galvo scanner and converging lens (e.g., as shown inFIG. 7). The beam splitter 1030 is used to combine or direct theexcitation beam onto the first low NA infinite conjugate objective 1000to facilitate the oblique plane imaging.

The system of FIG. 10 also includes a 3D scanning sub-system that has agalvanometer mounted planar scan mirror 1035, a first converging lens1040, and a second converging lens 1045. A computing sub-systemassociated with the system includes a graphical user interface andsoftware that allows a user to control the galvanometer mounted planarscan mirror 1035. The 3D scanning sub-system also includes a translationstage 1050 to provide coarse placement of the sample. An illuminationplane 1055 to be imaged is also shown. Similar to the other embodiments,3D reconstruction can be performed by a stitching algorithm which isused to stitch large 3D scan tiles, in conjunction with an Affinetransformation which is used to obtain the correct geometricalorientation of the sample. The 3D reconstruction can be performed by alocal or remote computing sub-system that is in direct or indirectcommunication with the system. In one embodiment, the same computingsub-system used to control the galvanometer mounted planar scan mirror1035 can be used to perform the 3D reconstruction. In alternativeembodiments, the system of FIG. 10 may include fewer, additional, and/ordifferent components.

As shown in FIG. 10, the 3D scanning sub-system (i.e., the galvanometermounted planar scan mirror 1035, first converging lens 1040, and secondconverging lens 1045) is arranged such that the galvo rotation axis isat a back focal plane of both of the lenses. Further, the conjugateimage planes of both objective tube-lens pairs is matched with a frontfocal plane of the two converging lenses surrounding the galvanometermounted planar scan mirror 1035. This arrangement ensures that rotationof the galvo scanner leads to a tilt-invariant scanning of the obliqueillumination plane in the sample volume. This arrangement furtherenables a sample to be imaged without any physical movement of thetranslation stage 1050 or objectives. The 3D re-construction of animaged volume is similar to the previously described embodiments.However, this embodiment also allows for acquisition of multiple 3Dtiles via a combination of galvo scanner sweep and manual coarsemovement of the translation stage 1050 that includes the sample beingimaged. Such tiles could be stitched together and 3D Affine transformedto obtain a true perspective large-scale 3D reconstruction of thesample. This approach eliminates mechanical vibration associated withany translation stage as all the tiles are essentially acquired throughremote scanning of the oblique plane illumination beam.

FIG. 11 depicts a low NA lens based oblique plane illumination imagingsystem in accordance with the fourth illustrative embodiment. An imagingsub-system of the system includes a first low NA objective (andassociated tube lens) 1100, a second low NA objective (and associatedtube lens) 1105, a third low NA objective (and associated tube lens)1110, a diffusive screen 1115 for intermediate image projection, and astandard camera sensor 1120. The standard camera sensor 1120 can be ofany pixel size, form factor, etc.

An illumination sub-system of the fourth embodiment includes anexcitation unit 1125. The excitation unit 1125 can be a fluorescenceexcitation source such as a laser diode or laser. The illuminationsub-system can also include a beam expander, slit aperture, andcylindrical lens (e.g., as shown in FIGS. 6A and 6B), or alternatively agalvo scanner and converging lens (e.g., as shown in FIG. 7). In someembodiments, the illumination sub-system can also include a beamsplitter to combine or direct the excitation beam onto the first low NAobjective 1100 to facilitate the oblique plane imaging.

A 3D scanning sub-system of the fourth embodiment includes an imagerelay and scan unit 1130. The image relay and scan unit 1130 can includea galvanometer mounted planar scan mirror along with two converginglenses. A computing sub-system associated with the system can include agraphical user interface and software that allows a user to control thegalvanometer mounted planar scan mirror of the image relay and scan unit1130. The 3D scanning sub-system also includes a translation stage 1135to provide coarse placement of the sample being imaged. An illuminationplane 1140 to be imaged is also shown. Similar to the other embodiments,3D reconstruction can be performed by a stitching algorithm which isused to stitch large 3D scan tiles, in conjunction with an Affinetransformation which is used to obtain the correct geometricalorientation of the sample. The 3D reconstruction can be performed by alocal or remote computing sub-system that is in direct or indirectcommunication with the system. In one embodiment, the same computingsub-system used to control the galvanometer mounted planar scan mirrorcan be used to perform the 3D reconstruction. In alternativeembodiments, the system of FIG. 11 may include fewer, additional, and/ordifferent components.

In the first three embodiments herein, it is important to position theimage sensor close to the objective at the distal end. This places tworestrictions on the image sensor: 1) the form factor of the sensor hasto be small and 2) the pixel size of the sensor has to be small (i.e.,even for 0.3 NA objectives, the pixel size of image sensor becomes theresolution limiting factor). Most scientific image sensors do not meetthese restrictions, and hence it is desirable to relax theserestrictions on image sensor choice. The fourth embodiment describedwith reference to FIG. 11 does this by use of a very fine graindiffusing screen placed on the intermediate image plane, which used tobe image sensor in earlier embodiments. The diffusing screen functionslike a projection screen and allows for placement of a third sub-systemin the form of the third low NA objective 1110, associated tube lens,and the standard camera sensor 1120 to form a magnified image of theprojected scene. As such, all three of the microscope objectives can below NA lenses that are still able to image an oblique illuminationplane.

The use of a diffusive screen followed by a magnification system with astandard camera sensor can similarly be applied to any of the otherembodiments described herein. Moreover, the imaging quality with thisand earlier embodiments can be further improved by adding in-planemotion to the diffusive screen through a vibrator or motor. The in-planemotion of the diffusive screen averages out any surface irregularitiesof the diffuser.

FIG. 12 is a block diagram of a computing device 1200 in communicationwith a network 1235 in accordance with an illustrative embodiment. Thecomputing device 1200 can be a computing sub-system that is incorporatedinto or in communication with any of the imaging systems describedherein. The computing device 1200 includes a processor 1205, anoperating system 1210, a memory 1215, an input/output (I/O) system 1220,a network interface 1225, and an imaging application 1230. Inalternative embodiments, the computing device 1200 may include fewer,additional, and/or different components. The components of the computingdevice 1200 communicate with one another via one or more buses or anyother interconnect system. The computing device 1200 can be any type ofnetworked computing device such as a laptop computer, desktop computer,smart phone, dedicating imaging computing sub-system, etc.

The processor 1205 can be any type of computer processor known in theart, and can include a plurality of processors and/or a plurality ofprocessing cores. The processor 1205 can include a controller, amicrocontroller, an audio processor, a graphics processing unit, ahardware accelerator, a digital signal processor, etc. Additionally, theprocessor 1205 may be implemented as a complex instruction set computerprocessor, a reduced instruction set computer processor, an x86instruction set computer processor, etc. The processor is used to runthe operating system 1210, which can be any type of operating system.

The operating system 1210 is stored in the memory 1215, which is alsoused to store programs, algorithms, network and communications data,peripheral component data, the imaging application 1230, and otheroperating instructions. The memory 1215 can be one or more memorysystems that include various types of computer memory such as flashmemory, random access memory (RAM), dynamic (RAM), static (RAM), auniversal serial bus (USB) drive, an optical disk drive, a tape drive,an internal storage device, a non-volatile storage device, a hard diskdrive (HDD), a volatile storage device, etc.

The I/O system 1220 is the framework which enables users and peripheraldevices to interact with the computing device 1200. The I/O system 1220can include a mouse, a keyboard, one or more displays, a speaker, amicrophone, etc. that allow the user to interact with and control thecomputing device 1200. The I/O system 1220 also includes circuitry and abus structure to interface with peripheral computing devices such aspower sources, USB devices, peripheral component interconnect express(PCIe) devices, serial advanced technology attachment (SATA) devices,high definition multimedia interface (HDMI) devices, proprietaryconnection devices, etc. In an illustrative embodiment, the I/O system1220 presents an interface to the user such that the user is able tocontrol the galvo scanner in any of the imaging systems describedherein.

The network interface 1225 includes transceiver circuitry that allowsthe computing device to transmit and receive data to/from other devicessuch as remote computing systems, servers, websites, etc. The networkinterface 1225 also enables communication through the network 1235,which can be one or more communication networks. The network 1235 caninclude a cable network, a fiber network, a cellular network, a wi-finetwork, a landline telephone network, a microwave network, a satellitenetwork, etc. The network interface 1225 also includes circuitry toallow device-to-device communication such as Bluetooth® communication.

The imaging application 1230 can include software in the form ofcomputer-readable instructions which, upon execution by the processor1205, performs any of the various operations described herein such asreceiving data, running algorithms, solving equations/matrices,performing 3D reconstruction, etc. The imaging application 1230 canutilize the processor 1205 and/or the memory 1215 as discussed above. Inan alternative implementation, the imaging application 1230 can beremote or independent from the computing device 1200, but incommunication therewith.

FIG. 13 depicts a United States Air Force (USAF) resolution test targetimaged in brightfield illumination using the oblique plane illuminationimaging system of FIG. 8 in accordance with an illustrative embodiment.To perform the imaging, a low numerical aperture (NA 0.1) was used alongwith low magnification (4×) objectives. Two Nikon 4×, 0.1 NA objectiveswere used for the first low NA finite conjugate objective 800 and thesecond low NA finite conjugate objective 805 depicted in FIG. 8. TheUSAF 1951 resolution test target was used as the sample for resolutionestimates. A small pixel camera sensor was used as the small pixelcamera sensor 810 of FIG. 8. The USAF target was back illuminated with abroadband white light source and imaged on the camera sensor. Inalternative embodiments, different components may be used. The figureshows Group 6, element 6 lines to be clearly resolved, which indicatesbetter than 5 μm resolution. Further enhancement in resolution can beobtained by imaging with a combination of smaller pixel camera sensorand higher NA microscope objectives.

FIG. 14 depicts a USAF resolution test target imaged in brightfieldillumination using the oblique plane illumination imaging system of FIG.11 in accordance with an illustrative embodiment. To perform theimaging, a low numerical aperture (NA 0.1) was used along with lowmagnification (4×) objectives. Three Nikon 4×, 0.1 NA objectives wereused as the first low NA objective 1100, the second low NA objective1105, and the third low NA objective 1110 depicted in FIG. 11. The USAF1951 resolution test target was again used as the sample for resolutionestimates. A fine diffuser, made of opaque white glass was used as thediffusive screen 1115 of FIG. 11. The USAF target was back illuminatedwith a broadband white light source and imaged with a relay lens andcamera sensor. In alternative embodiments, different components may beused. This imaging was performed with a static diffuser, resulting inminor speckling irregularities in the image. However, the figure showsGroup 5, element 5 lines to be clearly resolved, indicating better than10 μm resolution. Greatly enhanced resolution can be acquired by imagingwith in-plane motion of the diffuser screen.

The word “illustrative” is used herein to mean serving as an example,instance, or illustration. Any aspect or design described herein as“illustrative” is not necessarily to be construed as preferred oradvantageous over other aspects or designs. Further, for the purposes ofthis disclosure and unless otherwise specified, “a” or “an” means “oneor more.”

The foregoing description of illustrative embodiments of the inventionhas been presented for purposes of illustration and of description. Itis not intended to be exhaustive or to limit the invention to theprecise form disclosed, and modifications and variations are possible inlight of the above teachings or may be acquired from practice of theinvention. The embodiments were chosen and described in order to explainthe principles of the invention and as practical applications of theinvention to enable one skilled in the art to utilize the invention invarious embodiments and with various modifications as suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto and theirequivalents.

What is claimed is:
 1. An imaging system comprising: a first finiteconjugate objective at a frontal end of the system; a second finiteconjugate objective at a distal end of the system; a beam splitting ormerging element positioned between the first finite conjugate objectiveand the second finite conjugate objective; an excitation unit configuredto direct an excitation beam into a sample positioned in front of thefirst finite conjugate objective, wherein the excitation beam is in theform of an excitation plane; and an image sensor positioned facing thesecond finite conjugate objective, wherein the image sensor lies in aconjugate plane of an excitation beam illumination plane at the frontalend of the system.
 2. The system of claim 1, wherein the first finiteconjugate objective and the second finite conjugate objective have anumerical aperture of 0.5 or less.
 3. The system of claim 1, wherein thebeam splitting or merging element comprises a cube beam splitter or aplate beam splitter with excitation and emission filters.
 4. The systemof claim 1, wherein the beam splitting or merging element comprises adichroic beam splitter which allows only a fluorescence emission beam topass through to the distal end of the system.
 5. The system of claim 1,wherein the excitation unit includes any combination of a laser or laserdiode, a beam expander, a slit aperture, and a cylindrical lens to shapethe excitation beam into a planar excitation beam.
 6. The system ofclaim 1, wherein the excitation unit includes any combination of alaser, a galvanometer mounted planar-mirror based scanner, and aconverging lens to form a planar excitation beam, and wherein theexcitation unit emits the excitation beam at an inclined angle such thatresultant illumination is an oblique plane, tilted with respect to aprincipal axis of the first finite conjugate objective.
 7. The system ofclaim 1, wherein a tilt angle of the excitation beam and the imagesensor are matched such that the image sensor is able to image an entireilluminated plane at the same time.
 8. The system of claim 1, furthercomprising a translation stage to hold the sample, wherein thetranslation stage moves the sample in a direction perpendicular to anaxis of the first finite conjugate objective such that the sample can bemoved to form an image from neighboring optical slices which can bestitched together to form a three-dimensional image of the sample. 9.The system of claim 1, further comprising a computing system configuredto apply an affine transformation on a stack of acquired images, whereinapplication of the affine transformation is based on a tilt angle of theexcitation plane.
 10. An imaging system comprising: a magnifying unitpositioned at a frontal end of the system, wherein the magnifying unitincludes a first infinity corrected objective and a first tube lens; ade-magnifying unit positioned at a distal end of the system, wherein thede-magnifying unit includes a second infinity corrected objective and asecond tube lens; a beam splitting or merging element positioned inbetween the first infinity corrected objective and the second infinitycorrected objective; an excitation unit configured to direct anexcitation beam into a sample positioned in front of the first infinitycorrected objective, wherein the excitation beam is in the form of anexcitation plane; and an image sensor positioned directly in front ofthe second infinity corrected objective such that the image sensor liesin a conjugate plane of the excitation plane.
 11. The system of claim10, wherein the beam splitting or merging element is positioned betweenthe first infinity corrected objective and the first tube lens.
 12. Thesystem of claim 10, wherein the beam splitting or merging element ispositioned between the first tube lens and the second tube lens.
 13. Thesystem of claim 10, wherein the beam splitting or merging element ispositioned between the second tube lens and the second infinitycorrected objective.
 14. The system of claim 10, wherein the excitationunit emits the excitation beam either from an off-axis position or froman inclined position such that resultant illumination is an obliqueplane that is tilted with respect to a principal axis of the firstinfinity corrected objective.
 15. The system of claim 10, wherein tiltangles of the excitation plane and the image sensor are matched suchthat the image sensor can image an entire portion of the excitationplane at the same time.
 16. The system of claim 10, wherein the imagesensor comprises a two-dimensional (2D) light detector array whichacquires 2D images either through a global shutter mechanism or througha rolling shutter mechanism while the image sensor is staticallypositioned.
 17. An imaging system comprising: a first set of opticalelements including a first infinity corrected objective positioned at afrontal end of the system, a beam splitting or merging unit, an imagerelay and scan unit, and a second infinity corrected objectivepositioned at a distal end of the system; a second set of opticalelements including a third infinity corrected objective, a tube lens,and an image sensor; a diffusive screen positioned between the first setof optical elements and the second set of optical elements; and anexcitation unit configured to emit an excitation beam into a samplepositioned along an axis of the beam splitting or merging unit, whereinthe excitation beam is in the form of a planar excitation beam.
 18. Thesystem of claim 17, wherein the image relay and scan unit includes agalvanometer mounted planar mirror positioned between two converginglenses such that an axis of rotation of the galvanometer mounted planarmirror exactly matches with focal planes of the two converging lenses.19. The system of claim 17, wherein the diffusive screen acts as aprojection screen and is made of scattering particles, wherein a size ofthe scattering particles either smaller than a resolving power of thesystem or is comparable to the resolving power of the system.
 20. Thesystem of claim 17, wherein the diffusive screen is mounted on a movingrotor or vibration motor which allows for in-plane motion of thescattering particles to average out random particle images that overlayimages of the sample.